Support for Cultivating Biological Material

ABSTRACT

The present invention relates to a container ( 1 ) for cultivating biological material, comprising a solid inert substrate ( 3 ), a growth medium and biological material arranged in or on the substrate ( 3 ), wherein the container ( 1 ) is substantially closed, wherein the volume of the container ( 1 ) is sterile and wherein the biological material comprises sowing material, and to a method and the use of the container ( 1 ) for cultivating biological material.

The present invention relates to a container and method for cultivatingbiological material as according to the preamble of claims 1 and 10respectively, and to the use of the container as according to thepreamble of claim 12.

A container for cultivating biological material according to thepreamble of claim 1 is known from U.S. Pat. No. 6,032,409.

In the cultivation of biological material it is important to obtain thehighest possible yield. In the case of vegetable material the procedureis generally as follows. Firstly, biological cell material is placed ina first closed, sterile container. This container is generally providedwith an agar medium. This first container is placed under conditionssuch that the biological cell material starts to grow. Once thebiological cell material has begun to grow to some extent, the cellmaterial is removed from the agar situated in the first container andtransferred to a second container. During this transfer the cellmaterial is washed often so that substantially all the agar is removed.In the case of a large number of plant types the biological cellmaterial is then processed into so-called sowing material. Usualprocessing methods here are shredding or singulating, although otherprocessing methods are also possible. The thus obtained sowing materialis then placed in a second container, which container also comprises anagar medium. Once the sowing material has developed sufficiently, thesowing material is removed from the second container and arranged on orin a solid inert substrate situated in a third container. The developedsowing material is here generally washed with water such thatpractically all the agar is removed. This final step of removing andwashing the developed sowing material takes place manually and therebyhas a number of drawbacks. Firstly, the transfer of the developed sowingmaterial is very labour-intensive, which has an unfavourable effect onthe cost price of the biological material. Contamination of the sowingmaterial or the developed sowing material with micro-organisms furtheroccurs very easily. This results in the biological material not growing,or growing very poorly. This also has an unfavourable effect on the costprice. In addition, agar medium often still remains on the developedsowing material despite the washing. Finally, when this sowing materialis then arranged (manually) on or in a solid substrate, there is a highrisk of micro-organisms such as fungi multiplying easily in thisresidual agar medium. After the container with the solid inertsubstrate, as a final step the grown sowing material is transferredtogether with the substrate from this container and into a finalcontainer or pot provided with potting compost or the like. Thecultivation of biological material thus consists of diverse processingsteps and is herein a labour-intensive and relatively expensiveprocedure.

The present invention has for its object to provide a solution to theabove stated problems.

A first aspect of the present invention results in a container asaccording to claim 1.

The advantage of such a container is that the penultimate step oftransferring the developed sowing material from a second container to athird container with a solid inert substrate no longer has to takeplace. The above described operations such as the transfer and washinghereby no longer need be carried out. This has a favourable effect onthe cost price. Contamination of the sowing material withmicro-organisms is further also avoided.

The sowing material is preferably chosen from the group comprising seedssuch as vegetable seeds of for instance tomato, cucumber and paprika,seeds of ornamental plants such as Gerbera, Anthurium, Bromelia, Orchidand Spathiphyllum, and seeds of arable crops, shredded and singulatedvegetable material, vegetable somatic embryos, fern spores, adventive oraxillary shoots or parts thereof. The shredded vegetable materialpreferably comprises shredded fern parts, ferns, saintpaulia, primulaand lily. The adventive or axillary shoots or parts thereof preferablyoriginate from vegetable crops such as tomato, cucumber and paprika.

The adventive or axillary shoots or parts thereof further preferablyoriginate from ornamental plants such as Gerbera, Anthurium, Bromelia,Orchid, Spathiphyllum, fern, Syngonium, lily, or from arable crops suchas potato. Vegetable somatic embryos preferably used as sowing materialare somatic embryos of trees, such as for instance those of coniferoustrees (spruce, pine). The present invention is particularly importantfor this group of sowing material since these are susceptible toinfections with undesirable micro-organisms. It is therefore alsorecommended that the sowing material is sterile. It is noted here thatsterile is understood to mean not only an absolute sterility, but also adegree of sterility such as is necessary to allow the sowing material togrow.

The solid inert substrate preferably comprises rockwool, glass wool,coconut fibre, peat, hemp fibres, purane foam, potting compost and/orcellulose wadding or similar substrate. The advantage of thesesubstrates is that they are particularly suitable for cultivatingbiological material due to their inert properties. In addition,rockwool, glass wool, purane foam and cellulose wadding in particularcan be easily sterilized.

The growth medium preferably comprises water, salts (minerals) and/orsugars, such as an agar medium and preferably a liquid or (highly)diluted agar medium. Especially when the CO₂ concentration inside thecontainer is relatively high, there are preferably no, or only few,sugars present in the growth medium. The advantage hereof is that theautotrophic growth of the sowing material is encouraged, whereby itultimately becomes stronger.

At least one side of the container is preferably closed by means of asemi-permeable foil. The advantage hereof is that the container isclosed off from the environment, whereby a so-called micro-environmentis created. The advantage of such an environment is that the conditionsinside the container can be controlled very precisely. Thesemi-permeable foil plays an important part here, since it can be chosensuch that it is for instance permeable to determined gases but not toothers (for instance permeable to O₂ to the outside but not to CO₂). Inthis manner the conditions inside the space of the container can thus beset such that they are ideal for the biological material forcultivating, such as sowing material.

The volume of the container is preferably provided with a gas mixturewith an increased CO₂ concentration. The advantage hereof is that theautotrophic growth of the biological material is enhanced.

It is further advantageous when the container is provided with a valvefor administering a gas mixture, nutrients and/or growth-stimulatingagent, such as a hormone, to the volume of the container. Byincorporating a valve in the container the space inside the containercan be adjusted to the ideal conditions of the biological material atthat moment.

It is further recommended that the sowing material is situated in acarrier preparation. It is preferably situated in a gel, such as a gelon agar basis, in a liquid, likewise for instance on agar basis. Theadvantage hereof is that the first stage of the growth of the sowingmaterial takes place in the carrier preparation and a subsequent stagetakes place in the solid substrate located under the carrierpreparation. A further great advantage of the use of a carrierpreparation is that the sowing material can be arranged on the solidinert substrate in simple and fully automated manner. This has afavourable effect on the cost price of the biological material, as wellas the chance of contamination by micro-organisms. In addition to acarrier substance, the carrier preparation preferably comprisesdifferent ingredients which are necessary or advantageous for the growthof the sowing material. Examples of such ingredients are water, salts,sugars and seaweed. The growth medium added to the inert substratepreferably comprises water, salts (minerals) and sugars. It isrecommended here that the content of sugars in the inert substrate islower than in the carrier preparation.

As stated, it is recommended that the carrier preparation comprisesagar. The advantage hereof is that it is a very stable medium and thatdifferent ingredients such as water, sugars and salts (minerals) canreadily be added thereto.

The container is preferably an outer container in which at least onesub-container is placed, wherein the sub-container receives the solidinert substrate with sowing material. The advantage of such a containeris that the sub-containers can be provided separately of the containerwith the inert substrate, the growth medium and the sowing material. Itis thus possible to use less complex machines. An additional advantageis that, if one of the sub-containers were to be contaminated with anundesirable micro-organism, it does not spread so quickly over all theother containers. The container preferably comprises between 1 and 500sub-containers. A second aspect of the present invention relates to amethod for cultivating biological material, comprising the steps of:

-   -   providing a sterile biological cell material;    -   placing the cell material in a first closed, sterile container        provided with an agar medium and allowing the cell material to        grow in this first container;    -   removing the grown cell material from the first container and        dividing thereof into smaller parts to form sowing material,    -   placing the sowing material in a second container for further        growth, characterized by    -   arranging the sowing material in or on an inert solid substrate,        wherein the substrate is accommodated by the second container,    -   arranging a growth medium in the second container, and    -   closing the second container in sterile manner.

The advantage of the above described method is that the sowing materialno longer has to be cultivated first in a second container with an agarmedium and then in yet another container with a solid inert substratetherein. According to the invention these two steps take placesimultaneously in a container according to the invention. The advantagehereof is that the chance of contamination of the sowing material isreduced. In addition, the developed sowing material no longer has to beremoved from the container with agar and washed manually. This has afavourable effect on the cost price of the cultivated biologicalmaterial.

It is further recommended that, after growing, at least a part of thesolid inert substrate with further grown (developed) sowing material istransplanted into a container with a substantially equivalent substrate.The advantage hereof is that the so-called capillary action, i.e. theextraction of moisture and nutrients from the substrate on which thesowing material has developed, is avoided. This has a favourable effecton the quality of the cultivated biological material.

A final aspect of the present invention relates to the use of the abovedescribed container in the cultivation of biological material.

The invention will now be described further using the following drawingsand examples. These figures and examples serve only by way ofillustration of the invention and are in no way intended to limit thescope of the invention.

In the drawings:

FIG. 1 shows a schematic side view of a container according to theinvention;

FIG. 2 shows a schematic side view of a container according to theinvention provided with a plurality of sub-containers.

FIG. 1 shows a side view of a container 1 according to the invention.Within the walls 2 of container 1 is placed an inert solid substrate 3which is provided with growth medium. A quantity of sowing material incarrier material 4 is arranged on the top side of substrate 3. When thesowing material begins to develop, carrier material 4 is used up and thematerial continues to grow in substrate 3. Substrate 3 can then beseparated from the container and be transferred into a pot with pottingsoil or similar substrate.

FIG. 2 shows a side view of a container 1 according to the invention.Container 1 is provided with a plurality of sub-containers 6 placed incontainer 1. An inert substrate 3 is arranged in each sub-container 6. Acarrier preparation 4 with sowing material therein is arranged onsubstrate 3. Container 1 is closed along side walls 2 by means oftransparent plastic material. The top side of container 1 is closedusing a semi-permeable foil 5. This foil 5 ensures that the gascomposition of inner space 7 of container 1 has the desired compositionand humidity. Foil 5 further ensures that the sowing material does notbecome contaminated with undesired micro-organisms. To enable the gascomposition of inner space 7 of container 1 to be changed a valve 8 isplaced against one of the walls 2 of container 1. additional growthmedium can however also be added to substrate 3 of sub-containers 6through this valve 8.

EXAMPLE 1

In a first group 104 sub-containers were placed under sterile conditionsin a container according to the invention. The sub-containers weremutually connected on the top side such that they formed a tray.Arranged in the sub-containers was rockwool which was provided with agrowth medium. Sowing material in a carrier preparation was arranged onthe rockwool. The carrier preparation here comprised agar-agar, water,salts and sugar. The sowing material comprised shredded fern parts ofthe type Nephrolepis exaltata ‘Corditas’. The container, thesub-containers, the rockwool and the carrier preparation with sowingmaterial were all sterile. The container was then closed under sterileconditions using a semi-permeable foil (PET 12/PE30). The container withthe sowing material therein was incubated for 40 days at 25° C. and atan interval of 16 hours daylight and 8 hours night. The plants wereincubated until they were large enough to be transferred into aso-called transplant tray with potting soil. In this transplant traythey were placed in a greenhouse and kept there until they were largeenough to be placed in pots.

In a second group a large number (104) of sterile sub-containers wereprovided with an agar mixture on which sowing material was arranged(shredded fern parts of the type Nephrolepis exaltata ‘Corditas’). Thesub-containers were placed in a sterile container which was closed onone side using a semi-permeable foil (PET 12/PE30). The whole was thenincubated in controlled conditions, i.e. for 49 days at 25° C. and aday-night interval of respectively 16 and 8 hours. Once the sowingmaterial had formed sufficient roots, the sowing material wastransferred into a so-called transplant tray with potting soil. In thistransplant tray they were placed in a greenhouse and kept there untilthey were large enough to be placed in pots.

Table 1 shows the growth of the plants in a sterile container until theycan be transferred to a transplant tray with potting soil.

Table 2 shows the growth of the plants in a non-sterile transplant trayup to the moment the plants can be placed in pots.

Vitro-growth time is understood to mean the time required for the sowingmaterial to grow into a processable plant. Plant size is understood tomean the diameter of the plant at the top. Plant height designates theheight of the plant. Root length is understood to mean the length of theroots in the rockwool or in the agar. Rooting time is understood to meanthe time necessary to grow into a plant which can be potted. Finally,failure percentage indicates the percentage of sowing material whichdoes not develop into an acceptable plant. TABLE 1 Growth up to transferto transplant tray Group 1 Group 2 vitro growth time 40 days 49 daysvitro plant size 15 mm 11 mm vitro plant height 15 mm 10 mm vitro rootlength 8 mm 3 mm

TABLE 2 Growth up to transfer to pot Group 1 Group 2 rooting time 26days 34 days vivo plant size 130 mm 70 mm vivo plant height 50 mm 30 mmvivo root length 80 mm 50 mm failure percentage 0% 5%

Table 1 shows clearly that the plants from group 2 need more time togrow before they can be placed in pots. It is further shown that theroots of the plants of group 1 are better developed.

Table 2 clearly shows that the rooting time in the transplant tray withpotting soil is markedly shorter in the plants of group 1. This tablefurther also shows that the failure percentage of the second group ismuch higher than that of the first group.

1.-12. (canceled)
 13. A container for cultivating biological material,comprising: a solid inert substrate; a growth medium; and a biologicalmaterial in contact with the substrate, characterized in that thecontainer is substantially closed, and the container content is sterile.14. The container as claimed in claim 13, characterized in that at leastone side of the container is closed with a semi-permeable foil.
 15. Thecontainer as claimed in claim 14, further comprising: a gas mixture inthe container having an increased CO₂ concentration such thatautotrophic growth of the biological material is enhanced.
 16. Thecontainer as claimed in claim 15, further comprising: a valve for addingmaterial to the container.
 17. The container of claim 13, furthercomprising: a material chosen from a gas mixture, nutrient and agrowth-stimulating agent.
 18. The container as claimed in claim 13,further comprising: a carrier with the sowing materials dispersedtherein.
 19. The container as claimed in claim 18, characterized in thatthe sowing material is chosen from the group comprising seeds, shreddedor singulated vegetable material, vegetable somatic embryos, fernspores, adventive or axillary shoots or parts thereof.
 20. The containeras claimed in claim 13, characterized in that the solid inert substrateis selected from the group consisting of rockwool, glass wool, coconutfibre, purane foam, peat, hemp fibres, potting compost and cellulosewadding.
 21. The container as claimed in claim 13, further comprising:sub-container inside the container, wherein the sub-container receivessubstrate and sowing material.
 22. The container as claimed in claim 1,further comprising: between 1 and 500 sub-containers in the container.23. A method for cultivating biological material, comprising the stepsof: providing sterile biological cell material; placing the cellmaterial in a first closed, sterile container provided with an agarmedium; and allowing the cell material to grow in the first container;removing grown cell material from the first container; dividing the cellgrown material into smaller parts to form sowing material; placing asmaller part of the sowing material in a second container for furthergrowth; arranging the sowing material in contact with an inert solidsubstrate, wherein the substrate is accommodated by the secondcontainer; arranging a growth medium in the second container; andclosing the second container in sterile manner.
 24. The method asclaimed in claim 23, characterized in that after growth at least a partof the substrate with further grown sowing material is transplanted intoa container with a substantially equivalent or identical substrate. 25.A container for cultivating biological material, comprising: a solidinert substrate; a growth medium; and a biological material in contactwith the substrate, a gas mixture in the container having an increasedCO₂ concentration such that autotrophic growth of the biologicalmaterial is enhanced, characterized in that at least one side of thecontainer is closed with a semi-permeable foil and the container contentis sterile.
 26. The container as claimed in claim 25, furthercomprising: a valve for adding material to the container; a materialchosen from a gas mixture, nutrient and a growth-stimulating agent; anda carrier with the sowing materials dispersed therein.
 27. The containeras claimed in claim 26, characterized in that the sowing material ischosen from the group comprising seeds, shredded or singulated vegetablematerial, vegetable somatic embryos, fern spores, adventive or axillaryshoots or parts thereof.